Fertilization is one of the most fundamental processes in nature, yet critical gaps exist in our understanding of this essential process. One of the earliest and most prevalent barriers to successful reproduction is the fertilization of an egg by more than one sperm, or polyspermy. This common problem, faced by the eggs of all sexually reproducing species, causes severe chromosomal defects and leads to embryonic mortality. This project will investigate the molecular mechanisms that ensure that each egg is fertilized by only one sperm, thus allowing for normal embryonic development. In the eggs of many organisms, fertilization evokes a prolonged membrane depolarization, which acts as a fast block to polyspermy. The fast polyspermy block requires the activity of one of more ion channel, but the molecular identity of any required channel is not known. In many species, including frogs, Cl- channels likely mediates this process. Coincidentally, a fertilization-induced increase in Ca2+ also occurs prior to the fast polyspermy block. A possible connection between these two events is the recently identified Ca2+- activated Cl- channel encoded by the TMEM16a gene.
In specific aim one, I will identify the source of Ca2+ required for the depolarization at fertilization. Experiments outlined in specific aim two will uncover the role of the TMEM16a channel in the fast polyspermy block. Along with a fertilization-evoked increase in Ca2+, fertilization is also accompanied by a two-fold increase in phosphatidylinositol 4,5-bisphosphate (PIP2). The role that this elevated PIP2 may play in the first minutes after fertilization is unknown. Because PIP2 is a known regulator of structurally diverse ion channels and because the fertilization- evoked PIP2 elevation occurs within the time frame of the fast polyspermy block, I hypothesize that PIP2 regulates the fertilization-evoked depolarization. I will test this hypothesis in specific aim three and determine if PIP2 depletion affects the polyspermy block. The results of these experiments will contribute to our understanding of the biology of fertilization, and will provide the basis for future advances is reproductive medicine.

Public Health Relevance

Fertilization is one of the most fundamental processes in nature, yet many of the processes involved in this process are unknown. Using cutting-edge experimental techniques, this project will uncover the molecular mechanisms that ensure that each egg is fertilized by only one sperm. These findings will contribute to our understanding of the events that begin new life.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Transition Award (R00)
Project #
4R00HD069410-03
Application #
8913352
Study Section
Special Emphasis Panel (NSS)
Program Officer
Moss, Stuart B
Project Start
2012-09-01
Project End
2017-08-31
Budget Start
2014-09-03
Budget End
2015-08-31
Support Year
3
Fiscal Year
2014
Total Cost
$248,999
Indirect Cost
$49,599
Name
University of Pittsburgh
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213